CRISPR/Cas9: Transfection Application

What is CRISPR?

CRISPR, which stands for Clustered Regularly Interspersed Short Palindromic Repeats, refers to a set of DNA sequences in bacteria acquired from viruses that help the bacteria destroy viral DNA. The short sequences, as their name suggests, are palindromic, and are paired with the Cas9 endonuclease to cut apart foreign DNA if a bacteria is attacked by a virus. The CRISPR/Cas9 system can be modified for genetic modification purposes by selecting the specific segment of double-stranded DNA that is targeted by the Cas9 protein. If a gene-knockout is desired, then the Cas9 protein can be used to cut double-stranded DNA at two locations, after which the cell’s non-homologous end joining (NHEJ) DNA-repairing mechanism results in the elimination of the target gene from the DNA. Adding sequences to double-stranded DNA is also possible by having the Cas9 cleave a single strand of DNA at two locations. The additionally introduced sequence is designed so that it overlaps the sticky ends of the cleavage, and the cell’s homology-directed repair (HDR) mechanism will incorporate the sequence into the double-stranded DNA.

Structure of the CRISPR/Cas9 complex

The CRISPR/Cas9 system cuts DNA at points determined by a sequence that is attached to the Cas9 protein. This sequence, called crRNA, can be chosen in a laboratory setting and modified to fit specific needs. crRNA is connected to the Cas9 protein by tracrRNA, and may be combined with tracrRNA to form sgRNA (single guide RNA). A repair template may also be introduced to guide a cell’s DNA repair mechanism and introduce a specific gene into its DNA. The Cas9 protein is connected to a short, unmodifiable PAM (protospacer adjacent motif) sequence that determines the location at which Cas9 cleaves DNA. The PAM sequence can usually be found every ten or so base pairs in a DNA strand, but nonetheless sgRNA and repair templates must be designed to accommodate for the specific PAM sequence.

Applications in Transfection

Delivering the CRISPR/Cas9 into a cell is most efficiently done with viruses, but may also be accomplished through plasmid-delivery and lipofection. Cationic lipid-based delivery of the Cas9 protein with attached sgRNA has also been shown to be effective in delivering the protein complex into a cell. Altogen has developed reagent kits to aid in the process of both delivering and constructing a desired CRISPR/Cas9 complex. Purified reagents are used to structure sgRNA with a desired sequence, after which it is connected to the Cas9 protein and encapsulated in a lipid bilayer. The resulting transfection complex is then introduced into cells and results in either the removal of the targeted gene or the addition of a gene if desired.

In Vivo Transfection Reagents from Altogen Biosystems

RNAi has been used for in vivo target validation studies using animal models. The major challenge in performing RNAi studies in vivo is the effective, directed delivery of functional small RNA molecules into specific tissues. Altogen® In Vivo Transfection Reagents could be conjugated with siRNA (or microRNA) and administered intratumorally (i.t) or systemically via intravenous (i.v) tail vein injection in order to provide directed gene silencing in specific tissues, including liver, pancreas, kidney, and tumors. Selective knockdown could be seen as early as 24 hours after injection.